Q: Can this be used with cells other than HUVECs?
A: This assay can be used with any endothelial cells.
Q: What is the composition of the ECM gel solution?
A: The ECM gel solution is the same as matrigel, which is an ECM extract prepared from Engelbreth-Holm-Swarm (EHS) sarcoma produced in mice and contains laminin as a major component as well as collage type IV, heparan sulfate proteoglycan, entactin, and other minor components. It does contain growth factors but the levels are unknown.
Q: Are angiogenesis mediators required for this assay?
A: Angiogenesis mediators are not essential for this assay because tubes will form without the addition of any mediator. Because tubes will form without additional mediators, this assay is a good system for studying inhibition of tube formation but more difficult for studying enhancement, unless performing a time course. We do not include any mediators with this assay, but if used, the optimal concentration for the compounds being used should be determined. The key to getting good tube formation is to use enough healthy HUVEC per well, such as 50,000 cells/well. Typically more cells per well will result in better tube formation than fewer cells.
Q: How can I get better tube formation?
A: Here are recommendations to improve tube formation:
1. Increase the incubation time to 12h or overnight. The best tube formation occurs after an overnight incubation.
2. The ECM gel should be completely thawed before using. The gel can be thawed at 4C for 24 hours and flick the tube gently when ready to use, just before aliquoting into the wells. The gel will need 30 minutes to 1 hour to form, however the plate can be incubated longer than one hour at 37C to complete gel formation if necessary. It is important that the gel is completely solidified before using, because if it is semisolid the cells won’t be in the same dimension and there will be less cell to cell interaction, reducing tube formation.
3. Once the cells are added, the plate should not be moved as this can disturb tube formation.
Q: How can I prevent tubes from concentrating at the edges of the wells?
A: Tube formation on the edges is likely a result of a higher concentration of cells distributed around the edges of the well. This indicates that there are not enough cells in the well and the cell number should be increased.
Q: Can you recommend inhibitory angiogenesis agents?
A: Examples of inhibitor agents are:
1. MMP inhibitor: GM6001
2. PI3K inhibitor: Wortmannin
Q: How are tubes counted?
A: Results are usually reported as tubes/field, branch points/field or average tube length.